The capability to determine soon after fertilization, with nearly 100% accuracy, the sex of mammalian embryos would provide numerous advantages in the dairy and livestock industries as well as veterinary and human medicine. The economic efficiency of livestock and dairy operations would be significantly improved by allowing gestation to continue beyond the very early stage only for embryos of the desired sex. Certain diseases, such as X-chromosome-linked diseases in humans and comparable diseases in other mammals, affect individuals of only one sex; an early, nearly certain determination of the sex of an embryo which, if carried to term, might be an individual with such a disease would provide valuable information on which to base a decision whether or not to carry the embryo to term.
In situations where, for economic or health reasons, a determination of embryo sex is indicated, it is advantageous to determine the sex as soon as possible after fertilization. Risks, to the life and health of a female carrying an embryo, of having the embryo aborted increase substantially as the period of gestation lengthens. With livestock, it is economically inefficient, both because of risks to life and health and because of reduced reproductive efficiency, for a female to carry longer than necessary an embryo that ultimately will be aborted.
With recent advances in in vitro fertilization, long-term embryo-preservation and embryo-transfer technologies, it would be feasible to avoid all risks and costs associated with pregnancy and abortion of an embryo of undesired sex, if it were possible to determine highly accurately the sex of an in vitro fertilized or transferred embryo prior to or at the time transfer for gestation to term is carried out. See, e.g., King and Picard, "Sexing Cattle Embryos: An Update," in Proc. Ann. Conf. on Artificial Insemination and Embryo-Transfer, 1986, pp. 35-39.
Prior to the present invention, a sufficiently sensitive, rapid and reliable method, employing nucleic acid hybridization probes, for determining the sex of a mammalian embryo of fewer than about 10.sup.4 cells in three or fewer days has not been available. Thus, until the instant invention, it has not been possible, employing nucleic acid probes, to realize the many health and economic benefits of sexing embryos, with near certainty, soon after fertilization.
Human fetuses have been sexed, at 6 to 15 weeks gestational age, by nucleic acid hybridization of DNA from cells obtained from chorion or placental biopsy or embryonic fluid. In these fetal sex determinations, probes were used that correspond to DNA segments that occur about 10.sup.3 times in male human DNA and much more frequently in male than female human DNA. Vergnaud et al., Patent Cooperation Treaty Application Publication No. 86/00342; Vergnaud et al., Brit. Med. J. 289, 73-76 (1984); Lau et al., The Lancet, Jan. 7, 1984, pp. 14-16; Gosden et al., The Lancet, Dec. 25, 1982, pp. 1416-1419; Bostock et al., Nature 272, 324 (1978). Probes of the present invention, which are sufficiently sensitive to sex a mammal with DNA from fewer than 10.sup.3 of its cells, can also be used to sex fetuses.
DNA segments that preferentially hybridize to male, in comparison with female, human DNA have been found. See Kunkel et al., Science 191, 1189-1190 (1976); Cooke, Nature 262, 182 (1976); Bostock et al., supra; Gosden et al., supra; and Bishop et al., Nature 303, 831 (1983). The sequences of these segments are not known. The extent to which any of these segments preferentially hybridizes to male (in comparison with female) DNA of non-human species has not been tested and, hence, remains unknown. There is no segment that is known to bind preferentially to male, in comparison with female, DNA of species of genera Bos (bovine), Capra (caprine), Equus (equine), Ovis (ovine), and Sus (porcine).
Further, there is nothing in the prior art to indicate that any DNA segments exist in mammalian male DNA that could be used to provide the basis for a nucleic acid probe to sex by nucleic acid hybridization, in less than a few 3 days and with nearly 100% accuracy, a mammalian embryo at an early stage, at or before the time of transfer of the embryo for gestation to term.